DM mellitus (DM) is a common disorder with a prevalence of 15.5 million sufferers in the United States (1). In 2007, complications arising from DM cost the US healthcare system $58 billion (1). One major complication of DM is neuropathies. Constipation is a key consequence of enteric neuropathy and is significantly more common in DM patients with a prevalence of 60% compared to 15% in the general population. DM-associated enteric neuropathy results from hyperglycemia-induced neuronal apoptosis. However, the mechanism by which hyperglycemia drives neuronal apoptosis in DM remains enigmatic. In light of the increasing appreciation of the role of toll-like receptors (TLR) in driving a variety of chronic disease processes, especially those associated with oxidative stress and advanced glycation products (AGEs), which is known to result from hyperglycemia, we hypothesized that TLRs play a role in DM-associated enteric neuropathy. In support of this notion, we have observed that upregulation of one particular TLR, namely TLR4 is associated with hyperglycemia-induced neuronal apoptosis. Moreover, genetic and pharmacological ablation of TLR4 prevents both hyperglycemia-induced neuronal apoptosis and DM-associated colonic dysmotility. Thus, we hypothesize that hyperglycemia-induced activation of TLR4 results in neuronal apoptosis and, consequently, drives the colonic dysmotility associated with DM. To test the hypothesis and further investigate the underlying mechanism(s) of hyperglycemia-induced neuronal apoptosis, we will perform experiments to establish the role of TLR4 in mediating hyperglycemia-induced enteric neuronal apoptosis and elucidate the mechanism involved. Using primary enteric neurons from WT/TLR4-/- mice and knock down/overexpression strategies in the enteric neuronal cell line developed in our laboratory, we will establish the necessity and sufficiency of TLR4 in modulating hyperglycemia-induced enteric neuronal damage. The mechanism of hyperglycemia induced TLR4 activation will be examined focusing on the role of Advanced Glycation end products (AGES) and oxidative stress. Further, we will characterize the signaling events involved in hyperglycemia-induced TLR4 activation in enteric neuronal apoptosis focusing on the downstream targets of TLR4 including NF-:B. Finally, we will determine the role of TLR4 on enteric neuronal apoptosis and colonic dysmotility in vivo. Our preliminary data indicate that streptozotocin (STZ) -induced DM results in enteric neuronal apoptosis and loss of mouse colonic myenteric neurons. These changes are ameliorated in TLR4-/- DM mice. Taken together these studies will not only elucidate a novel mechanism involving TLR4 in the pathogenesis of hyperglycemia-induced enteric neuronal apoptosis but also provide "proof of principle" for targeted therapies that could be used for the treatment or prevention of dysmotility associated with DM. PUBLIC HEALTH RELEVANCE: The etiology and pathogenesis of the gastrointestinal complications of diabetes is largely unknown and thus treatment of this debilitating condition is a challenge to clinicians. In many patients the duration of diabetes correlates with the development of diabetic autonomic neuropathy as well as the presence of diabetic gastrointestinal complications such as diabetic constipation. In this proposal we will examine the mechanism of how hyperglycemia leads to loss of enteric neurons. Experiments outlined in this proposal will not only contribute to the understanding of the changes in enteric neurons in diabetes, but may also lead to new therapeutic targets for the altered gastrointestinal motility seen in diabetes.